Table 2 - uploaded by James Lewis Crooks
Content may be subject to copyright.
Bulk hybridomas and their immunizing antigen

Bulk hybridomas and their immunizing antigen

Source publication
Article
Full-text available
Significance The evolutionary hypothesis for T-cell antigen receptor–peptide major histocompatibility complex (TCR–pMHC) interaction posits the existence of germ-line–encoded rules by which the TCR is biased toward recognition of the MHC. Understanding these rules is important for our knowledge of how to manipulate this important interaction at the...

Contexts in source publication

Context 1
... we devised a system in which the responses of many dif- ferent T cells to antigen bound to the mutant MHCIIs could be assessed simultaneously. C57BL/6 mice were immunized separately with five different antigens (Table 2). Seven days later, T cells from the draining lymph nodes of the immunized mice were restimulated with their cognate antigens, expanded in vitro, and fused in bulk to the TCR αβ − BW5147 thymoma cell line to create T-cell hybrid- omas. ...
Context 2
... days later, T cells from the draining lymph nodes of the immunized mice were restimulated with their cognate antigens, expanded in vitro, and fused in bulk to the TCR αβ − BW5147 thymoma cell line to create T-cell hybrid- omas. The preparations were named for their target MHC-II allele, I-A b , and antigen ( Table 2). ...
Context 3
... were eluted from these preparations and subjected to MS or MS-MS analysis as previously reported (32) and as described in Materials and Methods. A preliminary MS-MS analysis of the peptides isolated from WT and mutant I-A b showed that they had I-A b - binding motifs (Table S2) (33,34). This finding served to vali- date our method of peptide isolation and suggested that the I-A b mutations did not affect the I-A b peptide-binding motif. ...

Citations

... For example, the associations between the genetic variability of MHC-I and the expression profiles of V-genes of TCRs of CD8 + lymphocytes are more significant in comparison with similar associations for CD4 + cells [124]. This is explained not only by the processes of coevolution [40], but also by closer contact between the V regions of the β-chain of TCRs of CD8 + cells and complementary regions of MHC-I molecules, which is determined by the difference in the spatial organization of TCRs of CD4 + and CD8 + lymphocytes [125][126][127]. This difference is established during maturation in the thymus, when the choice between CD4 and CD8 is determined by the TCR affinity for the corresponding class of MHC molecules [128]. ...
Article
Full-text available
The interaction of the T-cell receptor (TCR) with a peptide in the major histocompatibility complex (pMHC) plays a central role in the adaptive immunity of higher chordates. Due to the high specificity and sensitivity of this process, the immune system quickly recognizes and efficiently responds to the appearance of foreign and altered self-antigens. This is important for ensuring anti-infectious and antitumor immunity, in addition to maintaining self-tolerance. The most common parameter used for assessing the specificity of TCR-pMHC interaction is affinity. This thermodynamic characteristic is widely used not only in various theoretical aspects, but also in practice, for example, in the engineering of various T-cell products with a chimeric (CAR-T) or artificial (TCR-engineered T-cell) antigen receptor. However, increasing data reveal the fact that, in addition to the thermodynamic component, the specificity of antigen recognition is based on the kinetics and mechanics of the process, having even greater influence on the selectivity of the process and T lymphocyte activation than affinity. Therefore, the kinetic and mechanical aspects of antigen recognition should be taken into account when designing artificial antigen receptors, especially those that recognize antigens in the MHC complex. This review describes the current understanding of the nature of the TCR-pMHC interaction, in addition to the thermodynamic, kinetic, and mechanical principles underlying the specificity and high sensitivity of this interaction.
... This confirms the hypothesis that allele variants of MHC-I and MHC-II play an important role in the formation of TCR repertoires of CD4 + and CD8 + , respectively. Besides, it was shown that mutations in the conservative regions of MHC-I and MHC-II that contact with the complementary regions of TCRs influence the profiles of expression of TRAV and TRBV in the CD4 + and CD8 + repertoires and change their clonal organization (136,137). ...
Article
Full-text available
In this review, we described the structure and organization of antigen-recognizing repertoires of B and T cells from the standpoint of modern immunology. We summarized the latest advances in bioinformatics analysis of sequencing data from T and B cell repertoires and also presented contemporary ideas about the mechanisms of clonal diversity formation at different stages of organism development. At the same time, we focused on the importance of the allelic variants of the HLA genes and spectra of presented antigens for the formation of T-cell receptors (TCR) landscapes. The main idea of this review is that immune equilibrium and proper functioning of immunity are highly dependent on the interaction between the recognition and the presentation landscapes of antigens. Certain changes in these landscapes can occur during life, which can affect the protective function of adaptive immunity. We described some mechanisms associated with these changes, for example, the conversion of effector cells into regulatory cells and vice versa due to the trans-differentiation or bystander effect, changes in the clonal organization of the general TCR repertoire due to homeostatic proliferation or aging, and the background for the altered presentation of some antigens due to SNP mutations of MHC, or the alteration of the presenting antigens due to post-translational modifications. The authors suggest that such alterations can lead to an increase in the risk of the development of oncological and autoimmune diseases and influence the sensitivity of the organism to different infectious agents.
... Nonsynonymous amino acid substitutions within the binding groove of an MHC-II molecule would be predicted to profoundly affect CDR3 repertoires. Such substitutions may alter the surface of an MHC-II molecule involved in interaction with the TCR, the conformation of antigenic peptides, and the whole repertoire of presented peptides, thus affecting TCR binding and T cell activation (24,25). ...
... Previously, it has been reported that TRBV and TRAV segment usage is strongly associated with the expressed allelic variant of MHC-II (19,55,56). The observation that point substitutions in conserved stretches of amino acids in MHC-II alter TRBV and TRAV segment usage in CD4 cells but do not affect the CD4 + numerical count (25) provides further, direct evidence for preselection of TCR repertoires in MHC recognition. ...
... Convincing data show that the interplay of the TCR repertoire with certain MHC-II allelic variants can predispose individuals to autoimmune diseases (7). The individual MHC context determines the selection of CD4 + T cells and shapes the TCR repertoire, influencing both V and J segment usage (19,25) and frequencies of particular TCR CDR3 variants (60). ...
Article
T cell maturation and activation depend upon T cell receptor (TCR) interactions with a wide variety of antigenic peptides displayed in a given major histocompatibility complex (MHC) context. Complementarity-determining region 3 (CDR3) is the most variable part of the TCRα and -β chains, which govern interactions with peptide–MHC complexes. However, it remains unclear how the CDR3 landscape is shaped by individual MHC context during thymic selection of naïve T cells. We established two mouse strains carrying distinct allelic variants of H2-A and analyzed thymic and peripheral production and TCR repertoires of naïve conventional CD4 ⁺ T (T conv ) and naïve regulatory CD4 ⁺ T (T reg ) cells. Compared with tuberculosis-resistant C57BL/6 (H2-A b ) mice, the tuberculosis-susceptible H2-A j mice had fewer CD4 ⁺ T cells of both subsets in the thymus. In the periphery, this deficiency was only apparent for T conv and was compensated for by peripheral reconstitution for T reg . We show that H2-A j favors selection of a narrower and more convergent repertoire with more hydrophobic and strongly interacting amino acid residues in the middle of CDR3α and CDR3β, suggesting more stringent selection against a narrower peptide–MHC-II context. H2-A j and H2-A b mice have prominent reciprocal differences in CDR3α and CDR3β features, probably reflecting distinct modes of TCR fitting to MHC-II variants. These data reveal the mechanics and extent of how MHC-II shapes the naïve CD4 ⁺ T cell CDR3 landscape, which essentially defines adaptive response to infections and self-antigens.
... Moreover, the importance of evolutionarily conserved residues in the MHCII molecule was tested by introducing amino acid substitutions into either the a (A64Q) or b chain (R70A and T77A) of MHCII in mice. These mutations resulted in minor shifts in the TRAV and TRBV usage of positively selected T cells, but surprisingly did not have any effect on the frequency or diversity of thymocytes that were positively selected (80). ...
Article
From the initial observations by Zinkernagel and Doherty over 40 years ago of the need for T cells to recognize "altered self," our understanding of TCR recognition of peptide+ MHC has made significant advances. However, alongside a more detailed understanding of the interaction comes additional questions around precisely why T cells must limit themselves to MHC, when a greater range of ligand binding is demonstrably possible, and mechanistically, how MHC restriction achieves the necessary T cell survival and activation signals. The answer may well lie in the study of noncanonical or poorly signaling TCRs to understand the absolute requirements for effective TCR-pMHC recognition, continued advances in structural biology providing resolution of multimolecular complexes, and cryo-EM providing information on the dynamics of molecular localization and organization before and after TCR ligation of pMHC. © Philip G. Stevenson, 2019; Published by Mary Ann Liebert, Inc. 2019.
... More recently, we identified residues within the CDR2β loop that were both evolutionarily conserved and critical for efficient selection by MHC molecules (21,22). Conversely, mutating conserved residues on the α-helices of MHC molecules altered the peripheral TCR repertoire, as only the TCRs capable of compensating for the absence of these interactions were selected (23). Other studies have also corroborated the predisposition of TCR molecules to interact with pMHC complexes (24)(25)(26)(27). ...
Article
Full-text available
Significance Central to adaptive immunity is the interaction between the αβ T cell antigen receptor (TCR) and peptides presented by the major histocompatibility complex (MHC) molecules. It is fundamental to understand any potential generalities regarding a TCR-pMHC recognition event. Two theories have been proposed to explain how TCR recognition of pMHC is specified: The germline-encoded theory, which proposes an evolutionary “hardwiring” of the TCR for recognition of MHC molecules through germline-encoded motifs, and the selection theory of TCR recognition, which suggests that TCR selection during development imposes the constraint of MHC recognition. Here, we show that TCRs have intrinsic specificity for MHC molecules, inherent to TCRαβ sequences, and enhanced but not determined by coreceptor expression.
... Notably, structures that composed of TRBV13 has been extensively studied in mouse. Complexes that include TRBV13-2 show that amino acids in its CDR2 loop react with related sites on the MHCII α1 helix despite various docking angles of the TCR, and TRBV13-1 CDR1 and CDR2 loops have more than one docking site on α1 helix and shifts according to docking positions (52). A dynamic interplay between TCR and MHC molecules has gathered more and more evidence. ...
Article
Full-text available
T cells recognize antigens as peptides bound to major histocompatibility complex (MHC) proteins through T cell receptors (TCRs) on their surface. To recognize a wide range of pathogens, each individual possesses a substantial number of TCRs with an extremely high degree of variability. It remains controversial whether germline-encoded TCR repertoire is shaped by MHC polymorphism and, if so, what is the preference between MHC genetic variants and TCR V gene compatibility. To investigate the “net” genetic association between MHC variations and TRBV genes, we applied quantitative trait locus (QTL) mapping to test the associations between MHC polymorphism and TCR β chain V (TRBV) genes usage using umbilical cord blood (UCB) samples of 201 Chinese newborns. We found TRBV gene and MHC loci that are predisposed to interact with one another differ from previous conclusions. The majority of MHC amino acid residues associated with the TRBV gene usage show spatial proximities in known structures of TCR-pMHC complexes. These results show for the first time that MHC variants bias TRBV gene usage in UCB of Chinese ancestry and indicate that germline-encoded contacts influence TCR-MHC interactions in intact T cell repertoires.
... For example, our previous study using animals from this colony showed a high between-individual variation in MHC genes 30 . Thymic selection, which depends upon individual MHC composition, may therefore be, at least to some extent, responsible for the observed between-individual differences in TCR repertoires [61][62][63] . ...
Article
Full-text available
In recent years, immune repertoire profiling with high-throughput sequencing (HTS) has advanced our understanding of adaptive immunity. However, fast progress in the field applied mostly to human and mouse research, with only few studies devoted to other model vertebrates. We present the first in-depth characterization of the T-cell receptor (TCR) repertoire in a non-model mammal (bank vole, Myodes glareolus), widely used in ecological and evolutionary research. We used RNA from spleens, 5′RACE and HTS to describe V and J segments of TCRβ, qualitatively characterize preferential V–J segment usage and CDR3 length distribution. Overall orthology to murine genes was preserved, with 11 J and 37 V genes found in voles (although 3 V genes lacked a close orthologue). Further, we implemented unique molecular identifiers for quantitative analysis of CDR3 repertoire with stringent error correction. A conservative, lower bound estimation of the TCRβ repertoire was similar to that found for mice (1.7–2.3 × 10^5 clonotypes). We hope that by providing an easy-to-follow molecular protocol and on-line bioinformatics tools that do not require reference sequences (AmpliTCR and AmpliCDR3), we will encourage HTS immune repertoire profiling in other non-model vertebrates, thus opening new research avenues in e.g. comparative immunology, ecology and evolutionary biology.
... To be sure that the TCR chains analyzed in our experimental mice were actually those involved in positive selection of the cells bearing them, we crossed the DOWT or DO48A transgenic, TCR-/-mice with TCR-/-TCR-/-animals of each MHC haplotype to generate animals that were DOWT or DO48A transgenic, TCR-/-, TCR+/-. Naïve CD4 T cells were isolated from the lymph nodes of these animals and cDNA coding for their TCRs were sequenced as previously described (Silberman et al., 2016). PCR and sequencing errors in the germ line encoded portions of these sequences were corrected as described in the Materials and Methods section. ...
... RNA was isolated from purified naïve CD4 T cells, PCR'd to expand TCRa sequences and sequenced as described in (Silberman et al., 2016). Post-sequencing analysis was performed to identify the TRAV and TRAJ genes for each sequence along with its corresponding CDR3. ...
Preprint
Full-text available
Mature T cells bearing αβ T cell receptors react with foreign antigens bound to alleles of major histocompatibility complex proteins (MHC) that they were exposed to during their development in the thymus, a phenomenon known as positive selection. The structural basis for positive selection has long been debated. Here, using mice expressing one of two different T cell receptor β chains and various MHC alleles, we show that positive selection-induced MHC bias of T cell receptors is affected both by the germline encoded elements of the T cell receptor α and β chain and, surprisingly, dramatically affected by the non germ line encoded CDR3 of the T cell receptor α chain. Thus, in addition to determining specificity for antigen, the non germline encoded elements of T cell receptors may help the proteins cope with the extremely polymorphic nature of major histocompatibility complex products within the species.
Article
Niemann‐Pick Disease, type C1 (NPC1) is a fatal, autosomal recessive, neurodegenerative disorder caused by mutations in the NPC1 gene. As a result, there is accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system. This abnormal accumulation results in a cascade of pathophysiological events including progressive, cerebellar neurodegeneration, among others. While significant progress has been made to better understand NPC1, the downstream effects of cholesterol storage and the major mechanisms that drive neurodegeneration remain unclear. In the current study, we (i) implemented the use of a commercial, highly efficient standard flow‐ESI platform for protein biomarker identification and (ii) identified and evaluated protein biomarkers at a terminal time point in the NPC1 null mouse model. In this study, we observed alterations in proteins related to fatty acid homeostasis, calcium binding and regulation, lysosomal regulation, inositol biosynthesis and metabolism as well as signaling by Rho family GTPases. New observations from this study include altered expression of Pcp2 and Limp2 in Npc1 mutant mice relative to control, with Pcp2 exhibiting multiple isoforms and specific to the cerebella. This study provides valuable insight to pathways altered in the late‐stage pathophysiology of NPC1. This article is protected by copyright. All rights reserved
Article
T cell discrimination of self and non-self is predicated on αβ T cell receptor (TCR) co-recognition of peptides presented by MHC molecules. Over the past 20 years, structurally focused investigations into this MHC-restricted response have provided profound insights into T cell function. Simultaneously, two models of TCR recognition have emerged, centred on whether the TCR has, through evolution, acquired an intrinsic germline-encoded capacity for MHC recognition or whether MHC reactivity is conferred by developmental selection of TCRs. Here, we review the structural and functional data that pertain to these theories of TCR recognition, which indicate that it will be necessary to assimilate features of both models to fully account for the molecular drivers of this evolutionarily ancient interaction between the TCR and MHC molecules.